Mechanical stretch induces nerve sprouting in rat sympathetic neurocytes

Abstract

Sympathetic nerve sprouting (SNS) has been shown to occur after myocardial infarction (MI) and heart failure (HF) and is known to be responsible for the development of lethal arrhythmias. During MI or HF intracardiac cells are exposed to increased mechanical stretch. Molecular mechanisms which trigger sympathetic neural growth are largely unknown. Therefore, this study aimed to investigate the impact of mechanical stretch on rat neonatal sympathetic neurocytes of the superior cervical ganglion (SCG). Mechanical stretch resulted in an increased growth of sympathetic neurocytes. Furthermore, we could demonstrate that SCG neurocytes express nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), neurotrophin-3 (NT-3) and glial derived neurotrophic factor (GDNF) on mRNA and protein level. An increased NGF and CNTF expression, a down-regulated GDNF expression and an unchanged NT-3 expression were identified in the neurocyte cell culture supernatant of neurocytes exposed to mechanical stretch. However, neither brain derived neurotrophic factor (BDNF) mRNA and protein was expressed in SCG neurocytes, nor BDNF could be detected in the cell culture supernatant of SCG neurons. By anti-neurotrophin neutralizing experiments NGF and CNTF were identified as important stretch-induced growth-inducing factors. Losartan, an angiotensin-II type 1 receptor inhibitor, abolished the stretch-induced increase of NGF and CNTF expression and thereby prevented the stretch-induced neural growth. This study provides new molecular mechanisms by which the inhibitory effect of angiotensin-II type 1 receptor blockers on the neural/arrhythmogenic remodeling can be explained. However, further in-vivo studies are required to address this important issue.

Introduction

Sympathetic nerve sprouting (SNS) is a major factor for the development of malignant arrhythmias after myocardial infarction (MI) (Zhou et al., 1995) and congestive heart failure (HF) (Himura et al., 1993). Pathological outgrowth of sympathetic fibers and subsequent hyperinnervation seems to be an important contributor to an elevated sympathetic tone and has been reported to occur within the first hours of MI (Oh et al., 2006). Furthermore, SNS has been suggested to be a major contributor to sudden cardiac death (Cao et al., 2000a, Cao et al., 2000b). After MI increased nerve growth factor (NGF) expression has been shown to promote sympathetic hyperinnervation (Hasan et al., 2006), and may represent an adaptive mechanism by which the heart tries to maintain ventricular contractility at the cost of eventually triggered ventricular arrhythmias. There is evidence that inflammatory cells like myofibroblasts and macrophages contribute to the development of SNS in the ischemic injury area after MI (Hasan et al., 2006). If other contributing factors than inflammatory cells or NGF are involved in the pathogenesis of SNS still needs to be investigated. Mechanical stretch is such condition to which the cardiac cells are exposed after MI and during HF (Zimmerman et al., 2000, Ashikaga et al., 2005, Gao et al., 2007, Koba et al., 2008). Recently, we could demonstrate that mechanical stretch of ventricular cardiomyocytes induces a significant down-regulation of NGF (Rana et al., 2009). However, a stretch-induced NGF down-regulation in cardiomyocytes would not explain the induction of SNS after MI and HF. Because the myocardium in extensively innervated by the sympathetic nervous system and the impact of mechanical stretch on sympathetic neurocytes is unknown, this study aimed to investigate the impact of mechanical stretch on rat neonatal sympathetic neurocytes of the superior cervical ganglion (SCG). Furthermore, stretch-induced alterations of the neurotrophins NGF, neurotrophin-3 (NT-3), ciliary neurotrophic factor (CNTF), glial derived neurotrophic factor (GDNF) and brain derived neurotrophic factor (BDNF) were investigated. Mechanical stretch resulted in increased neural growth assessed by measuring the neurite outgrowth and growth associated protein 43 (GAP-43) expression. Furthermore, in the cell culture supernatant of neurocytes exposed to mechanical stretch NGF and CNTF expression was significantly increased, GDNF expression was down-regulated, NT-3 expression was unchanged, while BDNF could not be detected. By anti-neurotrophin neutralizing experiments, NGF and CNTF were identified as important stretch-induced neural growth-inducing factors. Angiotensin-II type 1 (AT-1) receptor blockade with losartan completely prevented the stretch-induced neural growth by attenuating the stretch-induced increase of NGF and CNTF.